Abstract

Comparative characteristics, common and distinctive features of qualitative and quantitative composition of myofibril proteins of skeletal muscles of some deep-sea fish species (Podonema longipes, Coryphaenoides cinereus, Coryphaenoides pectoralis) have been determined. Myofibrillar proteins were extracted from the skeletal muscle of three species deep-sea fish and their relative molecular mass was estimated by SDS/polyacrylamide gel electrophoresis. Subunit and quantitative compositions of deep-sea fish proteins were determined. The molecular weight of predominant contractile proteins, myosin (with heavy chains and two light chains), actin, troponin and tropomyosin was about 492, 47, 38 and 35 kDa, respectively for all fish species. The myosin/actin ratios were determined to be 2.85, 2.76 and 2.56 respectively for C. cinereus, P. longipes, and C. pectoralis. The Ca2+-ATPase activity of C. cinereus and P. longipes actomyosins was significantly higher at low ionic strengths (0.317 and 0.324 μM Pi mg-1 min-1 accordingly) than at high ionic strengths (0.257 and 0.221 μM Pi mg-1 min-1 accordingly). At the same time the Mg2+- ATPase activity value remained almost constant at both high and low ionic strengths (0.169–0.178 μM Pi mg-1 min-1). The Ca2+-ATPase activity of C. cinereus, P. longipes and C. pectoralis myosins was 0.534, 0.641 and 0.376 μM Pi mg-1 min-1 respectively.

Keywords

Actomyosin; ATPase; Deep-sea fishes; Myosin; Skeletal muscle tissue

Introduction

Deep-sea fish have not generally been used for food, but its
abundance suggests it could be an important source of highquality
protein. Deep-sea fish have much different significance for
industrial fishing. Three species of deep-sea fish Coryphaenoides
pectoralis, Coryphaenoides cinereus and Podonema longipes are
the most accessible for effective fishing in the Far-Eastern Seas of
Russia. These three species were chosen for investigations in the
present paper.

For processing of fish meat, the knowledge of the properties
of fish muscle proteins is necessary. The main aim of the present
paper was to determine the composition of basic myofibrillar
proteins and its ratio in skeletal muscle tissue.

Investigations of the biochemistry of muscle tissue of deepsea
fish have only incidental character and systematic study
of this has not been carried out. In most cases the studies of
researchers have been limited to mesopelagic species. So, there
are information about thermostability of deep-sea fishes skeletal
muscle actomyosin (Arai, K et al., 1973), myofibrillar proteins
as a whole (Uchiyama, H et al., 1978) and water soluble proteins
(Taguchi, T et al., 1981). But for all that, the information about
the basic and regulatory protein’s composition, of the skeletal
muscles of deep-sea fish species, is perfectly absent. Biochemical
properties of these proteins practically have not been studied.
At the same time, great amounts of information about these
species habits (Dayton, P.K and Hessler, R.R., 1972) and feeding
patterns (Drazen, J.C et al., 2001), along with their biological and
biochemical mechanisms of adaptation to low temperatures and
high pressures (Wakai, N et al., 2014; Morita, T., 2010) have been
accumulated in scientific literature at the present time.

The usual depth of the adults of these species range from
several hundred meters to upwards of 2,500 m. The tissue
composition of these deep-sea fish also suggests a midwater
lifestyle. The majority of deep-sea fish species are characterized
by high moisture contents in the muscle tissue (Somero, G.N.,
1992; Siebenaller, J.F et al., 1982). So, the white muscle tissue
of Coryphaenoides pectoralis is composed of approximately
92% water. This high value of muscle water content is likely an
adaptation to increase buoyancy without the metabolic costs of
buoyant lipids or a gas bladder (Drazen, J.C et al., 2001). The
compositions of the skeletal muscle myofibrillar proteins of these
species are unknown. Therefore, determining the composition of
these muscle proteins seems very important for understanding the
contribution of muscle myofibrillar proteins to meat texture.

Materials and Methods

Raw materials

White muscles (musculus lateralis magnus) of three species
of deep-sea fish Coryphaenoides pectoralis, Coryphaenoides
cinereus and Podonema longipes were used for research. All
fishes were immediately filleted and dorsal white muscles were
coarsely minced, mixed with an equal volume of glycerol, and the
mixture was frozen at –40°C and was kept frozen at –24ºC until
use, not longer than one month.

Preparation of proteins

Preparation of myofibrils isolation was carried out by method
of Kato (Kato, N., Uchiyama, H., Tsukamoto, S., Arai, K., 1977;
Kato, S., Konno, K., 1993). Actomyosins of deep-sea fish skeletal
muscles were isolated by the extraction at high ionic strength, pH
6.4. Actomyosins were purified twice by precipitating at ionic
strength 0.1 and recovered as solution in 0.6 M KCl. For additional
purification of actomyosins and removing the regulatory proteins
the method of fractional precipitation with ammonium sulfate was
used. So, pure actomyosins were isolated at ammonium sulfate
concentration below 35% of degree of saturation.

Myosins were isolated by the method of Bruggmann and Jenny
(Bruggmann, S and Jenny, E., 1975) with some modifications. The
solution of 0.3 M KCl, 10 mM Na4P2O7, 1 mM MgCl2 in 0.15
KH2PO4 (pH 6.4) was used as basic one for extraction and after
dilution with 10 volumes of water for “batch” process on DEAESepharose
4В (Pharmacia Biotech AB). So, myosins with highest
possible ATPase activity were prepared.

Tropomyosins were isolated by the method of Bailey (Bailey,
K., 1948). The isolation procedure of protein was carried out in
high ionic strength solutions (0.5-0.6 M) with using ammonium
sulfate as a salting-out agent. Troponins were isolated by the
method of Ebashi (Ebashi, S et al., 1968). Actin and actinine were
isolated by the method of Ishikawa (Ishikawa, H., 1983).

Polyacrylamide gels with acrylamide concentration 3.5%
of concentrating layer were used. Concentrating process was
carried out at 15 mA and 210 V. Gels with constant concentration
of acrylamide 10, 12.5 or 15% and gradient concentration of
acrylamide 10-15% were used as separating ones. The separating
process was carried out at 5 mA and 100 V. Disk gel was stained
with 0.15% coomassie brilliant blue R-250 in 50% methanol-10%
acetic acid at 25ºC for 5 min and was detained with 50%
methanol-5% acetic acid at 25ºC overnight.

Densitometry

Densitometry was carried out on the stained disk gel using a
chromatoscanner UV-260 (LKB, Sweden) at wavelength of 640
and 700 nm.

ATP- sensitivity was determined by the method of Weber
(Weber, H.H and Portzehl, H., 1952). The relative viscosity of
2-3 mg/ml protein solutions was determined before (ηrel) and after
(ηrel. ATP) addition of 1 mM ATP at 25ºC. The ATP- sensitivity was
calculated with using formula:

ATPsens=(lgηrel – lgηrel. ATP)·100/ ηrel. ATP

Superprecipitation was induced at 20ºС by adding 1 mM
ATP to the reaction mixture containing 60 mM KCl, 2 mM
MgCl2, 0.1 mM CaCl2, 20 mM Tris-maleate, (pH 6.8), 0.1 mg/
ml of actomyosin. Absorbance at 540 nm was recorded by UV-
1800 Shimadzy spectrophotometer. Extent of superprecipitation
was expressed as ΔO.D=Amax – A0, where Amax is the maximum
absorbance upon addition of ATP and A0 is the absorbance in
absence of ATP. The rate of super precipitation was expressed as
t1/2 – time corresponding half-cycle of process when (A-A0)=(Amax – A0)/2.

Statistical analysis

The results were presented as arithmetic means and their
standard deviations. The applied methods were evaluated
statistically with the use of STATISTICA 8.0 software.

Results and Discussion

Structure and characterization of actomyosin of
deep-sea fish skeletal muscles

Disk-gel electrophoretic patterns of myofibrillar proteins of three species of deep-sea fish obviously were not speciesspecific
(Figure 1). It is significant that the myosin/actin ratio
for actomyosins of deep-sea fish skeletal muscles is lower
in comparison with traditional vertebrates. The ratios were
determined to be 2.85, 2.76 and 2.56 respectively for C. cinereus,
P. longipes, and C. pectoralis. How actomyosins of deep-sea fishes
skeletal muscle hydrolyzed ATP and ATPase activity depended on
their Ca2+ and Mg2+ concentration. The Ca2+-ATPase activity of C.
cinereus and P. longipes actomyosins was significantly higher at
low ionic strengths (0.317 and 0.324 μM Pi mg-1 min-1 accordingly)
than at high ionic strengths (0.257 and 0.221 μM Pi mg-1 min-1 accordingly). At the same time the Mg2+- ATPase activity value
remained almost constant at both high and low ionic strengths
(0.169-0.178 μM Pi mg-1 min-1 There was not dependence on
actomyosin of C. рectoralis ATPase activity both effectors and
ionic strength. Actomyosins of all objects showed highest ATPase
activity at 5 mM Ca2+. The lowest values of ATPase activity were
shown when both effectors were used in equal concentration (2.5
mM). The results of cross effect studies on the added Ca2+ and
Mg2+ ions showed that the effect of Mg2+ overcomes that of Ca2+ in the testing conditions. In the presence of ATP, actomyosins of
the skeletal muscles dissociated in high ionic strength solutions. It
was illustrated in the changes of certain attributes such as viscosity
(Figure 2) and parameters of ultraviolet fluorescence (UVF).

As shown in Table 1, increases in the half-width of UVFsignals
occurred as a result of protein denaturation. However, the
wavelength of emissions remained stable during reaction with ATP, whereas the shift to the long-wavelength field of spectrum
occurred in cases denatured proteins. It is important to note that
changes of the UVF-spectrums characteristics were irreversible
ones during reaction with ATP, and were only restored after the
reprecipitation of proteins.

We have observed actomiosins aggregation under the influence
of heat treatments. The aggregation rates increased with increasing
temperatures, and denaturation changes became apparent at
temperatures of more than 35°C. By means of UVF (Turoverov,
K.K et al., 2002; Turoverov, K.K et al., 1999) we ascertained
that the degree of denaturation (as a partition of denaturated
protein) had a logarithmic dependence on heating time. At that,
the denaturation rate constant had an exponential dependence on temperature at a range of 35-55ºC (Figure 5).

Structure and characterization of myosin of
deep-sea fish skeletal muscles

The main problem of myosin extraction was the quantitative
and selective isolation of this protein from myofibrills. The 0.3
M KCl, 5 mM EDTA, 1 mM dithiothreitol solution, pH 6.4, was
used for the selective myosin extraction (Figure 6, Track 1). 10
mM sodium pyrophosphate when added to this solution increased
neither selectivity nor effectiveness in myosin extraction (Figure
6, Track 2). At the same time the addition of only 1 mM ATP and 2
mM MgCl2 to the extraction mixture allowed a remarkable increas
in effectiveness and a slight increase in selectivity (Figure 6, Track
3). And vice versa, a pretreatment of the concentrate myofibrils
with a low ionic strength solution, (Figure 6, Track 4) or in the
aggregate with an extraction using Mg-ATP (Figure 6, Track 5),
both allowed to maximum selectivity and poor effectiveness of
the extraction of myosin. Extraction was carried out at a ratio of
muscle tissue: extractive solvent, 1:5 for all variants.

The most satisfactory results of myosin’s isolation were
achieved by using the Bruggmann-Jenny method (Bruggmann, S.,
Jenny, E., 1975). The yields of myosin preparations were around
10-14 mg from every 100 g of muscle tissue. Myosin, prepared in
this method, showed all the typical properties of myosin already
shown in well-investigated cases: ATP hydrolysis activity, low
ATP-sensitivity, negative superprecipitation reaction, and low viscosity compared to those of actomyosin. The characteristics
of the myosins of deep-sea fish’s skeletal muscles are shown in Table 3.

Subunit and quantitative compositions of deep-sea fishes myosins were determined by mean of total and partial dissociation
(Table 4). The full separation of all light chains of myosins
occurred in low-concentrated urea solutions. It is necessary to
note that different concentrations of urea solutions were used to
examine the myosins treatment. So, the necessary and sufficient
urea concentration for the separation of C. cinereus myosin light
chains was 2.0 M, for P. longipes - 2.8 M and for C. pectoralis - 3.2
M. These values were determined by means of UV-fluorescence
(Figure 7). The point where the increase of half-width of UVFspectrum
signals began, during titration of myosin or actomyosin
solutions by urea, served as the criterion for the determination
of urea concentration. At that, the values of the A320/A360 ratio
remained more than 1. It signifies the absence of deep delocalized
changes in the myosin structure during such treatments.

The most effective results of total myosin light chains
separation were realized during brief (3-4 min) intensive heat
treatments (53°C) of protein solution. Most of the proteins (about
60%) were aggregated in these conditions (Figure 8, Track 2).
Actomyosin may be used as a substitute in this experiment, instead
of myosin. A partial light chain separation of myosin was carried
out as demonstrated, in Table 4 conditions.

Figure 8: Myosin dissociation in the different conditions. SDS-PAGE was performed on a 10-15% gradient acrylamide gel (Podonema longipes example is shown).
1. Myofibrillar proteins of Podonema longipes skeletal muscles.
2. Dissociation under the influence of 2.8 M urea and during minute heating of up to 53°С.
3. Dissociation during 60-minutes heating of up to 40°С.
4. Dissociation in 4.7 M ammonium chloride solution.
5. Dissociation under the influence of 5, 5’-dithio-bis-2-nitrobenzoic acid.

Heat treatments at 40°C and lasting less than 60 min
allowed us to selectively remove light chains LC1 and LC3 from
myosin (Figure 8, Track 3). The light chain LC3 was removed
from myosin by the prolonged incubation of the myosin in a
concentrated ammonium chloride solution (4.5-4.7 M) (Figure
8, Track 4). The treatment of myosin with 5, 5’-dithio-bis-2-
nitrobenzoic acid solution led to the removal of light chain LC2 only (Figure 8, Track 5).

Structure and characterization of actin of deepsea
fish skeletal muscles

Actin of skeletal muscle tissues of deep-sea fish has been
easily extracted by using low ionic strength solutions. However,
most regulatory proteins have been extracted in this manner as
well. So, the method, based on depolymerization of fibrillar actin
and on higher thermostability of actin globular form, was used
(Dominguez, R and Holmes, K.S., 2011). Extraction time was
limited to 5 minutes. Because upon the increase of extraction
time, the fair quantity that passed into extract impurity proteins
exceeded actinin quantity by more than 5 times. The actin and actinin were separated by the means of fractional precipitation.
The purity of the actin preparations was calculated, from the
area of the band in the densitogram pattern for SDS-PAGE, to be
90-94%. Their yields were 54-70% based on the quantity in the
muscle tissue.

Structure and characterization of tropomyosin
and troponin of deep-sea fish skeletal muscles

Tropomyosins of deep-sea skeletal muscles were isolated and
purified by the Bailey method (Bailey, K., 1948). Tropomyosin
is known to be a protein without tryptophane in its structure and
its UV-spectrum looks like a spectrum of tyrosine (Staprans, I.,
Watanabe, S., 1970). Proteins with identical spectral characteristics
were isolated by us, from deep-sea fishes myofibrils: UV-spectrum
maximum 277.4-278.8 nm, and AU278 / AU260 ratio-1.98-2.03.
However, different compositions of subunits were established
(Tables 5 and 6). Only in the case of C. pectoralis tropomyosin
the α:β ratio was normal according to the value which is known
for an overwhelming majority of vertebrates (Shiraishi, F., 1993). skeletal muscles of C. cinereus, P. longipes and C. pectoralis are
almost equal to each other (Table 6).

Troponins of deep-sea fishes muscle tissue were differed in
their subunits ratio too. In contrast to traditional objects (Shiraishi,
F., 1993; Yates, L.D and Greaser, M.L., 1983; Perry, S.V., 1998)
troponins of deep-sea fishes skeletal muscles aren’t equimolar The α:β ratio for C. cinereus and P. longipes tropomyosins was
below typical values. Molecular weights of tropomyosins of complexes. Prevalence of the heaviest component was typical
for all examined objects. The distribution of light components
was different though, but, on the whole, molecular weights of
troponins were close to each other (Table 7).

The non-equimolar composition of troponin was earlier known
(Islam, A., 2006) to have been observed. The different rates of
proteolytic degradation of troponin components are considered to
be the reason for the non-equimolar composition of troponin. The
processes of proteolysis of troponin components occurred both
during isolation of proteins from muscle tissue and during freeze
storage of intact muscle (Tanokura, M et al., 1983; Wei, B and
Jin, J.P., 2011). In our case, the troponin components ratio was
practically constant or slightly changed during either treatment
or storage of muscle tissue. The isolation of all basic protein
has allowed us to carry out precise references of signals of the
electrophoretic pattern to the relevant proteins (Figure 9) and to
calculate its abundance (Table 8).

These results indicated that the composition of proteins of
skeletal muscle tissue of examined deep-sea fish species differs in
contents of myosin, whereas other main myofibrillar proteins are
contained in comparable quantities. Consequently, the different
values in the myosin/actin ratio were observed.